PROJECT SUMMARY Aging is a recognized risk factor in peripheral vascular disease (PVD). A hallmark of aging in humans is loss of vascular function reflected, in part, by inhibited self-renewal of endothelial cells (EC). An unmet need is the emergence of therapies that stop or reverse endothelial senescence (decreased capacity for cellular division, self-renewal) and vascular insufficiency. A second pathognomonic feature of aging is increased reactive oxygen species (ROS). Pathologic ROS impair vascular flow, but the mechanisms responsible for these maladaptive age-related changes in the endothelium remain unknown. Thrombospondin-1 (TSP1) is increased in the plasma, arterial walls and interstitial fluid of peripheral vascular disease (PVD) patients and is linked to disease severity and loss of vascularity. We have found that with aging, wild type (WT) mice show induction of TSP1 and its cognate cell membrane receptor CD47 and this was associated with decreased vasodilator- mediated changes in blood flow, an effect not observed in TSP1-/- and CD47-/- mice. We also reported that TSP1, via CD47, potently stimulates NADPH oxidase 1 (Nox1)-derived ROS and impedes hind-limb blood flow. Preliminary data by our group show that TSP1 is upregulated in older adults, and it inhibits endothelial self- replication and promotes endothelial senescence in peripheral vessels. These findings led us to propose the overarching hypothesis that TSP1, via CD47 engagement and Nox1 activation, impairs self-renewal through exacerbated ROS production culminating in EC senescence and vasculopathy. Three specific aims will be pursued to: (1) test in vitro that with aging TSP1 induces Nox1-mediated suppression of key self- renewal genes and promotes endothelial cell (EC) senescence; (2) explore in vivo that inhibition of the TSP1-CD47 axis as well as attenuating its downstream Nox1 effector restores self-renewal, angiogenesis and perfusion in aged animals; and (3) investigate that perturbing the TSP1-CD47-Nox1 axis in aged human tissue ameliorates age-related endothelial and vascular dysfunction. The proposal will employ multiple molecular and genetic tools to interrogate mechanisms into TSP1-CD47-mediated activation of Nox1 in aging human and mouse vessel function, self-renewal and angiogenesis, as well as generate two EC-specific knockout mice to test our hypotheses. These studies will also provide robust pre- clinical data towards rational development of new and effective treatments for EC senescence and vasculopathy.